The free radical nitric oxide (NO) is a chemical messenger that is involved in regulating blood pressure, homeostasis, platelet aggregation, immuno-integrity and neurotransmission. NO is produced in many cell types, including endothelial cells, neurons, airway epithelial cells and macrophages. In blood vessels, NO mediates endothelium-dependent vasodilation. NO is also involved in maintaining basal vascular tone and regulating regional blood flow. In the nervous system NO plays a role in neurotransmission, synaptic plasticity, peristalsis, penile erection, neuro-degenerative disease, and excitotoxicity. In the immune system, NO is produced by activated macrophages and neutrophils as a cytotoxic agent against tumor cells and pathogens.
The formation of NO is catalyzed by a family of enzymes termed nitric oxide synthases. The nitric oxide synthases (NOSs) are encoded by three different genes. Two of the genes encode isoforms that bind calmodulin in a reversible Ca2+ dependent manner. These two isoforms are known as neuronal NOS (nNOS) or NOS I and endothelial NOS (eNOS) or NOS III. nNOS and eNOS are constitutively expressed. nNOS and eNOS have approximately 55% amino acid sequence identity. The third gene encodes an inducible isoform known as inducible NOS (iNOS) or NOS II. iNOS is constitutively expressed only in select tissues such as lung epithelium, and is more typically synthesized in response to inflammatory or pro-inflammatory mediators. Each NOS is comprised of an N-terminal oxygenase domain and a C-terminal reductase domain, with a Ca2+xe2x80x94 calmodulin (CaM) binding region of approximately 30 amino acids located between the two domains. (See FIG. 1.)
Recent studies have shown that in vivo gene transfer of polynucleotides encoding different NOS isoforms may represent a therapeutic strategy for diseases characterized by decreased bioavailability of NO, such as vascular diseases. Specifically, Lloyd-Jones and Bloch have shown that gene therapy employing an nNOS-expressing adenoviral vector increased the sensitivity of a normal rabbit""s carotid arteries to acetylcholine and also reversed the deficit in endothelium-dependent vascular relaxation in cholesterol-fed rabbits. (Lloyd-Jones and Bloch (1996) Annual Rev. Med. 47:365-75.) Quian et. al. showed that in vivo transfer of the nNOS gene into cholesterol-fed rabbits reduced vascular adhesion molecule expression, lipid deposition, and inflammatory cell infiltration in the carotid arteries of such animals. (Qian, et. al. (1999) Circulation 98, 2979-2982.) Von der Leyen et al have shown that in vivo transfer of an eNOS-expressing plasmid to balloon denuded rat carotids significantly limited the subsequent development of neointimal hyperplasia. The eNOS-expressing plasmids were delivered via a liposome-Sendai virus hemagglutinin protein complex. (Von der Leyen, et. al. (1995) PNAS 92:1137-1141.) Kullo et al showed that adenovirus-mediated transfer of the gene for eNOS to the adventitia of rabbit carotid arteries had a favorable effect on vascular reactivity (Kullo, et. al. (1996) Circulation 96:7, 2254-2261.) Shears et al have shown that in vivo transfer of an adenovirus expressing iNOS reduced vasculopathy in rat aortic allografts. (Shears, et. al. (1997) J. Clin. Invest. 100:8, 2035-2042.) Thus, all three major NOS isoforms appear to be reasonable candidates for in vivo applications to achieve or augment NO production.
Accordingly, it is desirable to have polynucleotides which encode NOS. It is especially desirable to have polynucleotides which encode NOS proteins or polypeptides that are fully active at all possible intracellular concentrations of O2.
The present invention provides polynucleotides which encode variants of NOS. The xe2x80x9cNOS variantxe2x80x9d comprises a substitution mutant, wherein the tryptophan that is normally located on the alpha 3 helix, six residues upstream from the cysteine which binds heme in the corresponding non-variant NOS, is replaced with one of the other 19 naturally-occurring amino acid residues. Preferably, the targeted tryptophan is replaced with an amino acid residue that forms no or weaker hydrogen bonds with the NOS heme thiolate.
The present invention also relates to vectors and recombinant cells comprising a nucleic acid which encodes a NOS variant. The present invention also relates to NOS variant proteins and polypeptides.